Abstract Heart failure and arrhythmias occur together, are marked by increased reactive oxygen species (ROS), and are major, unsolved public health problems. Despite this, ROS can be beneficial. However, clear molecular mechanisms supporting the benefits of ROS are lacking. The potential for ROS to be ?good and bad? is called the ROS paradox. This Outstanding Investigator Award application will propose innovative approaches to understanding and dissecting healthy from pathological ROS. The goal of this research program is to test a disruptive concept that ROS activation of the multifunctional calcium and calmodulin kinase II (CaMKII) by isoform and organelle selective pathways determine physiological and pathological outcomes of ROS signaling. We discovered that oxidation activates CaMKII (ox-CaMKII, Erickson Cell 2008) and established that ox-CaMKII?, the major CaMKII isoform in heart, causes heart failure and arrhythmias. Our newest studies extended these findings to show that ox-CaMKII? also contributes to asthma. In sharp contrast, our new preliminary data indicate that ox-CaMKII?, an isoform enriched in skeletal muscle, is beneficial and enhances endurance, insulin sensitivity, lean phenotype, PGC-1?, and expression or brain derived neurotrophic factor (BDNF), a myokine that improves exercise capacity and protects against myocardial injury. Our group discovered that CaMKII is present in mitochondria and that mitochondrial-targeted inhibition of CaMKII protects against common forms of myocardial injury associated with high ROS (Joiner Nature 2012). Here we propose to pursue new models of global (i.e. body-wide) mitochondrial CaMKII inhibition in flies (Drosophila melanogaster) and mice developed for the proposed OIA studies. Mitochondrial CaMKII inhibited flies have a prolonged lifespan and resistance to paraquat induced oxidant injury, while mice with global CaMKII inhibition have reduced mortality in sepsis and increased weight gain. We performed new phosphoproteomic and metabolic analyses that identified unanticipated mitochondrial CaMKII targets with central roles in metabolism and ROS production. Our preliminary computational modeling and experimental data suggest that physiological activation of mitochondrial CaMKII contributes to a beneficial metabolic fight or flight response that increases ATP; however, sustained and excessive mitochondrial CaMKII activity causes dilated cardiomyopathy due to loss of complex 1 and ATP deficiency. We and our collaborators will use state of the art computer modeling, imaging and metabolic studies, and generate a panel of mouse and Drosophila models using CRISPR/Cas9 to dissect contributions of CaMKII to specific targets relevant to heart failure, arrhythmias and to health. This research program requires an R35, or similar, mechanism because of its highly innovative concept that challenges current paradigms in CaMKII and ROS biology, the need to manage large amounts of data and generate new animals models, and the requirement for a prolonged timeframe.